Carotenoid cleavage dioxygenases (CCDs) are a unique family of non-heme iron oxygenases. The human genome encodes three family members, namely BCO1, BCO2, and RPE65 that are critical for carotenoid and retinoid metabolism related to vision. Thus, mutations in their genes are associated with blindness, vitamin A deficiency and altered carotenoid plasma levels. Emerging evidence indicates that these enzymes are targets for retinoid cycle modulators, a drug class that can correct ocular disease states related to retinoid metabolism. Here we present studies designed to unveil the biochemical and structural basis of the substrate specificity and catalytic mechanism of these important enzymes. Dissecting molecular mechanisms underlying CCDs' functions will advance our knowledge of biochemical processes that govern this disease-associated metabolism and will assist the design of therapeutics directed against ocular disease states. In Aim 1, we will determine the architecture of the enzyme/substrate complex at an atomic scale. Properly folded but enzymatically inactive metal-substituted CCDs will be crystallized in the presence and absence of bound substrate/inhibitors. Spectroscopy techniques will be used to survey the metalloprotein substrate arrangement in the active center. These analyses will provide a wealth of information about the oxygen activation process and changes in metal oxidation state during the reaction. Together with 18O labeling experiments, these studies will provide unprecedented insights into the catalytic mechanism of these enzymes. In Aim 2, we will unveil the biochemical and structural basis of vitamin A production. In this pathway, BCO1 and BCO2 must distinguish between more than 600 structurally related naturally occurring carotenoids. We will employ recombinant purified enzymes and natural and synthetic substrates to determine the selectivity of these enzymes for a specific ionone ring site of these bicyclic substrates. Information about the architecture of the protein-substrate complex will allow us to determine critical amino acid residues that account for the stereo- and region-specificity of CCDs. With the help of unique knockout mouse models we will translate this knowledge into the in vivo situation and study how deficiency of either CCD can affect ocular retinoid metabolism and functioning.